title: Metabolic re-programming by MALAT1 depletion in prostate cancer

city: Biella

first: Paola

last: Ostano

address: via Malta 3

country: Italy

institute: Fondazione Edo ed Elvo Tempia

summary: In this study, we sought to investigate the metabolic role of MALAT1, one of the most abundant cancer-associated long non-coding RNA, in prostate cancer. MALAT1 targeting by gapmerization reduced expression of some tricarboxylic acid cycle enzymes including the malic enzyme 3 and the pyruvate dehydrogenase kinases 1 and 3 as well as the choline kinase A. In consequence, prostate cancer metabolism switched toward a glycolytic phenotype characterized by increased lactate production paralleled by growth arrest, and cell death. Conversely, the function of mitochondrial succinate dehydrogenase and expression of oxphos enzymes was markedly reduced, suggesting for a decreased tricarboxylic acid cycle and mitochondrial respiration activity. Interestingly, a similar effect was observed in several prostate cancer-derived organotypic slice cultures, in which metabolism became more glycolytic and apoptotic. Based on these observations, we elaborated a predictive algorithm, in which those metabolic enzymes sensitive to MALAT1 targeting proven successfully to predict tumor recurrence in a subset of patients. In summary, MALAT1 targeting by gapmerization activates a metabolic switch in the prostate cancer cell and tumor tissue unraveling a role for crucial metabolic enzymes in tumor progression and outcome.

overall_design: Gene expression profiling of prostate cancer cell lines and prostate cancer-derived organotypic slice cultures with MALAT1 targeting by gapmerization reduced expression. For each cell line biological replicate and for each OSC (except for OSC#11 for which low amount of RNA was available), two independent hybridizations were carried out, with dye-swap labelling. PC3 cells with and without Malat1 depletion were hybridized three times, with a technical dye-swap replicate for one of the two independent biological replicates.

first: S

last: Nanni

first: A

last: Aiello

first: C

last: Salis

last: Re

last: Cencioni

first: L

last: Bacci

first: F

last: Pierconti

last: Pinto

last: Ripoli

first: P

last: Baroni

first: G

last: Lazzarino

first: B

last: Tavazzi

first: D

last: Pugliese

middle: F

last: Bassi

last: Grassi

last: Panunzi

last: Chiorino

last: Pontecorvi

last: Gaetano

last: Farsetti


title: Castration-induced SPARC down-regulation in stroma cells drives neuroendocrine differentiation of prostate cancer

status: Public on Jul 30 2021

last: Ostano

type: Expression profiling by high throughput sequencing

summary: Fatal neuroendocrine (NE) prostate cancer evolves from castration-resistant adenocarcinoma as a mechanism of resistance to androgen deprivation/anti androgen receptor therapies. Evidence suggests the microenvironment as a possible source of soluble factors mediating neuroendocrine differentiation (NED), but molecular mechanisms are unknown. Using a transgenic mouse model we show that upon castration tumor cells up-regulate GRP78, which triggers a cascade leading to down-regulation of the matricellular protein SPARC in the nearby stroma. SPARC loss allows stroma cells to release IL-6, a known inducer of NED. A drug targeting GRP78 blocks NED in castrated mice. These events find correlation in prostate cancer patients developing focal NED after androgen deprivation therapies. Our results candidate SPARC and GRP78 for diagnostic and therapeutic purpose, respectively.

overall_design: RNA-sequencing was performed on T23 adenocarcinoma cells, in different co-culture conditions. Samples were analyzed in triplicate.

first: Claudia

last: Enriquez

first: Valeria

last: Cancila

first: Renata

last: Ferri

first: Roberta

last: Sulsenti

first: Irene

last: Fischetti

first: Matteo

last: Milani

first: Ilaria

last: Gregarin

first: Maurizia

last: Mello-Grand

first: Enrico

last: Berrino

first: Marco

last: Bregni

first: Giuseppe

last: Renne

first: Claudio

last: Tripodo

first: Mario

middle: P

last: Colombo

first: Elena

last: Jachetti


title: The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation [MPRA]

city: Stanford

first: Daniel

middle: Sunwook

last: Kim

state: CA

address: 269 Campus Drive, CCSR 2150

country: USA

institute: Stanford School of Medicine

summary: A deep-learning framework interprets multi-omic data across epidermal differentiation, identifying cooperative DNA sequence rules that regulate gene modules. Massively parallel reporter analysis validates temporal dynamics and cis-regulatory logic.

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: Lentiviral MPRA of selected genomic constructs in primary keratinocyte differentiation with reporter RNA collected on days 0, 3, and 6

middle: S

first: David

last: Reynolds


title: The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation [HiChIP]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: HiChIP (Mumbach 2016) collected at days 0, 3, and 6 during in vitro keratinocyte differentiation

first: Namyoung

last: Jung

first: Laura

last: Donohue


title: A low Smc flux avoids collisions and facilitates chromosome organization in B. subtilis

city: Lausanne

first: Anna

last: Anchimiuk

state: Vaud

address: UNIL-Sorge District

department: Department of Fundamental Microbiology

country: Switzerland

institute: University of Lausanne

summary: SMC complexes are widely conserved ATP-powered loop extrusion motors indispensable for the faithful segregation of chromosomes during cell division. How SMC complexes translocate along DNA for loop extrusion and what happens when two complexes meet on the same DNA molecule is largely unknown. Revealing the origins and the consequences of SMC encounters is crucial for understanding the folding process not only of bacterial, but also of eukaryotic chromosomes. Here, we uncover several factors that influence bacterial chromosome organization by modulating the probability of such clashes. These factors include the number, the strength and the distribution of Smc loading sites, the residence time on the chromosome, the translocation rate, and the cellular abundance of Smc complexes. By studying various mutants, we show that these parameters are fine-tuned to reduce the frequency of encounters between Smc complexes, presumably as a risk mitigation strategy. Mild perturbations hamper chromosome organization by causing Smc collisions, implying that the cellular capacity to resolve them is rather limited. Altogether, we identify mechanisms that help to avoid Smc collisions and their resolution by Smc traversal or other potentially risky molecular transactions.

overall_design: Chromosome conformation capture and ChIP-seq of Bacillus subtilis wt cells and mutants

first: Virginia

last: Lioy

first: Frédéric

last: Boccard

first: Stephan

last: Gruber


title: Organ specific analysis of PIFq in Arabidopsis

city: Beijing

first: Lei

last: Wang

address: Nanxincun 20, Xiangshan, Haidian District

department: Key Laboratory of Plant Molecular Physiology

country: China

institute: insitute of botany, chinese academy of sciences

summary: The apical hook is the predominant organ to suffer the mechanical pressure and sense the first beam of light when protruding through the soil. In order to understand the organ specific mechanism of this phenomenon, we performed RNA-sequencing analysis using cotyledon, apical hook and hypocotyl organs of 4-day dark grown Col-0 and pifq mutant

overall_design: Totally 9 wild type samples and 9 pifq mutant samples, three organs and each has three replicates.

first: Yuanyuan

last: Zhang


title: Exercise during pregnancy prevents parental obesity-induced decline of metabolic function in adult offspring

city: Copenhagen

first: Ali

last: Altintas

address: Blegdamsvej 3B

department: NNF Center for Basic Metabolic Research

country: Denmark

institute: Copenhagen University

type: Methylation profiling by high throughput sequencing

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Microarray analysis of gene expression profile in Sjögren’s syndrome patients

status: Public on Jul 29 2021

city: Bethesda

first: Hongen

last: Yin

state: MD

address: 10 Center Dr. Bldg10, Rm1A21

institute: NIDCR

summary: EMT gene expression profile in Sjögren’s syndrome patients were determined by microarray analysis performed by Arraystar Inc. (Rockville, MD) using LncPath™ Human EMT Pathway Microarray (Cat# AS-LP-004H). N=219 potential coding targets related to the EMT signaling pathway were tested.

overall_design:
Total RNA was extracted from FFPE samples from labial salivary glands of N=20 Sjögren’s syndrome patients, and then were tested for EMT pathway microarray analysis according to the manufacturer’s protocol (Arraystar Inc.)

Contributor: Arraystar Inc. (Rockville, MD)

first: John

middle: A

last: Chiorini


title: Dendritic cells maintain anti-tumor immunity by positioning CD8 resident memory T cells in the skin through the CXCL16-CXCR6 axis

city: lebanon

first: Jennifer

last: Vella

address: 1 medical center drive

institute: Dartmouth College

summary: Tissue-resident memory (TRM) T cells are emerging as critical components of the immune response to cancer; yet, requirements for their ongoing function and maintenance remain unclear. Antigen presenting cells (APC) promote TRM cell differentiation and re-activation but have not been implicated in sustaining TRM cell responses. Here, we identified a novel role for dendritic cells in supporting tissue resident memory to melanoma. We showed that CD8 TRM cells remain in close proximity to dendritic cells in the skin. Depletion of CD11c+ cells results in rapid disaggregation and eventual loss of melanoma-specific TRM cells. Additionally, we determined that TRM migration and/or persistence requires chemotaxis and adhesion mediated by the CXCR6/CXCL16 axis. The interaction between CXCR6-expressing TRM cells and CXCL16-expressing APCs was found to be critical for sustaining TRM cell-mediated tumor protection. These findings substantially expand our knowledge of APC functions in tissue resident memory T cell homeostasis and longevity.

overall_design: comparing CD8 T cells, CD11c+CD11bneg and CD11c+CD11b+ monocytes from melanoma associated vitiligo skin of mice

middle: L

first: Aleksey

last: Molodtsov

first: Christina

middle: V

last: Angeles

first: Mary

middle: J

last: Turk

first: Yina

middle: H

last: Huang


title: Expression patterns of TnAV-6a1 genes in the combination of fat body, Epidermis and tracheal matrix tissues compared to hemolymph viral vesicles.

city: Riverside

first: Heba

middle: A. H. M.

last: Zaghloul

address: 900 University Ave

department: Entomology

institute: University of California, Riverside

summary: Purpose: To compare the TnAV-6a1 variant infected Trichoplusia ni larval transcriptome of hemolymph viral vesicles versus those of other major larval tissues specifically, the fat body, epidermis and tracheal matrix (referred to as FET tissues).

overall_design: Method: We used a TnAV-6a minor variant strain (referred to as TnAV-6a1) to infect third or early fourth instars Trichoplusia ni larvae. Then, collected the hemolymph viral vesicles and fat body, epidermis and tracheal matrix for transcriptomic studies.The TnAV-6a1 variant genomic DNA library prepared following manufacturer instructions of the NEBNext DNA Library Prep Master Mix Set for Illumina (New England, BioLabs). Thirty four contig sequences were generated using Trinity software with “genome guided” assembly option (Grabherr et al., 2011). For genome sequencing we used the Illumina MiSeq (2 x 300, Paired-end). For RNA-sequencing we used the HiSeq2500 Illumina sequencer (Noll Core Facility at the UC Riverside Institute for Integrative Genome Biology), in duplicate.

first: Robert

last: Hice

first: Peter

last: Arensburger

first: Brian

last: Federici


title: Differential gene expression analysis of genes deregulated in Ptch1+/+/Tis21 knockout  vs.  Ptch1+/+/Tis21 wild-type mice

city: Catania

first: Sebastiano

last: Cavallaro

address: Via Paolo Gaifami, 18

institute: IRIB-CNR

summary: We generated a new medulloblastoma model by crossing Patched1 heterozygous mice, which develop medulloblastomas with low frequency, with mice lacking the Tis21 gene. Double knock-out mice show a significant increase in medulloblastoma frequency and hyperplastic EGL lesions formed by preneoplastic GCPs. We found that Tis21 deletion does not affect the proliferation of GCPs but it inhibits their differentiation and their ability to migrate outside the EGL, remaining for a prolonged time in the cerebellum proliferative area. We identified as a drug target and major responsible of this migration defect the down-regulation of the promigratory chemokine Cxcl3 rather than a number of down-regulated tumor inhibitors and up-regulated tumor facilitators, and focusing on pathways potentially involved in the tumorigenesis and on putative new drug targets. Among them, we identified the activation of the PI3K/AKT/mTOR pathway that contributes to medulloblastoma development and to high frequency tumorigenesis when the Tis21 gene is down-regulated. Moreover, we used a novel PI3K inhibitor in nodules derived from primary Ptch1+/−/Tis21KO causing a significant reduction of tumor growth, inhibiting proliferation and increasing apoptosis.

overall_design: Two-condition experiment, cerebellar granule +precursor cells (GCPs) of Ptch1+/+/Tis21 knockout  vs.  Ptch1+/+/Tis21 wild-type mice isolated at P7, for each genotype were used three biological replicates.

first: Felice

last: Tirone


title: Identification of transcriptional difference between Imatinib-sensitive and resistant patients with gastrointerstinal stromal tumor (GIST)

city: Shanghai

first: Shengli

last: Li

address: 650 Xinsongjiang Road

institute: Shanghai Jiao Tong University School of Medicine

summary:
Purpose: The goals of this study are to characterize the transcriptional dysregulation of GIST. More importantly, the transcriptome differences betweeen Imatinib-sensitive and resistant patients are compared to identify RNAs that may impact Imatinib resistance.
Methods: High-throughput RNA sequencing (RNA-seq) was employed to capture the transcriptional changes in GIST compared to normal samples. RSEM was used to quantify gene expression and DESeq2 was utilized to compared expression changes between different sample groups.

overall_design: Tanscriptome-wide comparison of Imatinib-sensitive and resistant GIST patients

first: Junyi

last: He

first: Yong


title: TNFR2 signaling regulates the immunomodulatory function of oligodendrocyte precursor cells

status: Public on Jul 28 2021

city: Miami

first: Haritha

last: Desu

state: Florida

address: 1095 NW 14th Terrace

institute: University of Miami

summary: Multiple sclerosis (MS) is a neuroimmune disorder characterized by inflammation, CNS demyelination, and progressive neurodegeneration. Chronic MS patients exhibit impaired remyelination capacity, partly due to the changes that oligodendrocyte precursor cells (OPCs) undergo in response to the MS lesion environment. The cytokine tumor necrosis factor (TNF) is present in the MS affected CNS and has been implicated in disease pathophysiology. Of the two active forms of TNF, transmembrane (tmTNF) and soluble (solTNF), tmTNF signals via TNFR2 mediating protective and reparative effects, including remyelination, whereas solTNF signals predominantly via TNFR1 promoting neurotoxicity. To better understand the mechanisms underlying repair failure in MS, we investigated the cellular responses of OPCs to inflammatory exposure and the specific role of TNFR2 signaling in their modulation. Following treatment of cultured OPCs with IFNg, IL1b and TNF we observed by RNA sequencing marked inflammatory and immune activation of OPCs, accompanied by metabolic changes and dysregulation of their proliferation and differentiation programming. We also established the high likelihood of cell-cell interaction between OPCs and microglia in neuroinflammatory conditions, with OPCs able to produce chemokines that can recruit and activate microglia. Importantly, we showed that these functions are exacerbated when TNFR2 is ablated. Together, our data indicate that neuroinflammation leads OPCs to shift towards an immunomodulatory phenotype while diminishing their capacity to proliferate and differentiate, thus impairing their repair function. Furthermore, we demonstrated that TNFR2 plays a key role in this process, suggesting that boosting TNFR2 activation or its downstream signals could be an effective strategy to restore OPC reparative capacity in demyelinating disease.

overall_design: RNAseq of primary WT and TNFR2 KO oligodendrocyte precursor cells that were either not stimulated or stimulated for 3 hours with TNF, IFNgamma, and IL1beta

first: Roberta

last: Brambilla


title: Transcriptional profiling of basilar artery and basilar terminus intima and media in rabbits after bilateral carotid ligation

city: BUFFALO

first: Kerry

middle: E

last: Poppenberg

state: NY

address: 875 Ellicott St

institute: University at Buffalo

summary: New Zealand white rabbits underwent bilateral carotid ligation to increase flow in the basilar artery (BA) or sham surgery. 24 hours post-surgery, the BA and basilar terminus (BT) were embedded for sectioning. The intima and media were separately laser-microdissected from the sections, and whole transcriptome RNA-seq was performed on each tissue in order to more thoroughly characterize potential molecular signals between endothelium and smooth muscle during flow-induced arterial remodeling.

overall_design: Female New Zealand white rabbits underwent bilateral common carotid artery ligation surgery (n=2) or a sham surgery (n=2). After 24 hours, vascular sections of the basilar artery (n=4) and the basilar terminus (n=4) were embedded for sectioning. The intima (n=8 samples) and media (n=8 samples) were separately laser-microdissected from the sections. Whole transcriptome RNA-seq was performed on each tissue sample (n=16).

first: John

last: Kolega

first: Vincent

middle: M

last: Tutino

first: Hee

middle: W

last: Lim


title: Robust Three-dimensional (3D) Expansion of Bovine Intestinal Stem Cells: A Model for Replacement of Animal Experiments Toward Animal Welfare

city: Wanju-gun

first: Bo Ram

last: Lee

state: Jeollabuk-do

address: 1500, Kongjwipatjwi-ro, Iseo-myeon

department: Animal Biotechnology & Environment

country: South Korea

institute: National Institute of Animal Science

summary: we successfully developed bovine intestinal organoids representing typical crypt-villus and epithelium’s characteristics from bovine small intestinal (jejunum) crypts

overall_design: Examination of 4 different samples

first: Bo

middle: R


title: Lipidomic profiling of clinical prostate cancer reveals targetable alterations in membrane lipid composition

status: Public on Jul 27 2021

city: Bedford Park

first: Luke

last: Selth

state: SA

address: Flinders Drive

department: College of Medicine and Public Health

country: Australia

institute: Flinders University

summary: Dysregulated lipid metabolism is a prominent feature of prostate cancer that is driven by androgen receptor (AR) signaling. Here we used quantitative mass spectrometry to define the “lipidome” in prostate tumors with matched benign tissues (n=21), independent unmatched tissues (n=47), and primary prostate explants cultured with the clinical AR antagonist enzalutamide (n=43). Significant differences in lipid composition were detected and spatially visualized in tumors compared to matched benign samples. Notably, tumors featured higher proportions of monounsaturated lipids overall and elongated fatty acid chains in phosphatidylinositol and phosphatidylserine lipids. Significant associations between lipid profile and malignancy were validated in unmatched samples, and phospholipid composition was characteristically altered in patient tissues that responded to AR inhibition. Importantly, targeting tumor-related lipid features via inhibition of acetyl-CoA carboxylase 1 significantly reduced cellular proliferation and induced apoptosis in tissue explants (n=13). This first characterization of the prostate cancer lipidome in clinical tissues reveals enhanced fatty acid synthesis, desaturation and elongation as tumor-defining features, with potential for therapeutic targeting.

overall_design: RNA-seq in prostate primary tumour explants treated with enzalutamide or vehicle control.

first: L

last: Butler

first: J

last: Swinnen

first: D

last: Lynn


title: Expression profile of MESH1 and ATF4 silencing RCC4 cells

city: Durham

first: Jen-Tsan

middle: Ashley

last: Chi

state: NC

address: 101 Science Drive, DUMC 3382

institute: Duke University

summary: MESH1 encodes for Metazoan SpoT Homolog 1, which is a homologue of bacterial SpoT that mediates bacterial stringent response. In human cells, we found that MESH1-silencing induces an extensive transcriptional response in clear cell carcinoma cell line RCC4 that partially overlap with mammalian cell integrated stress response (ISR), which ATF4 up-regulation is an essential branch of the response. In this study, the goal is to elucidate the role of ATF4 up-regulation in MESH1-silencing transcriptional response.

overall_design:
Human clear cell renal carcinoma cell line RCC4 were transfected with siRNA targeting MESH1 (siMESH1), ATF4 (siATF4), or MESH1 with ATF4 together (siMESH1+siATF4), and compared with non-targeting siRNA control (siNT). All samples were prepared as biological triplicates. Total RNA was collected after 3 days of reverse transfection for cDNA synthesis. The influence of silencing MESH1 and ATF4 on gene expression was derived by zero transformation process, in which we compared the gene expression level with siMESH1, siATF4 or siMESH1+siATF4 to the average of gene expression level in control (siNT) samples.
biological replicate: siNT-1, siNT-2, siNT-3
biological replicate: siMESH1-1, siMESH1-2, siMESH1-3
biological replicate: siMESH1+siATF-1, siMESH1+siATF-2, siMESH1+siATF-3
biological replicate: siATF4-1, siATF4-2, siATF4-3

first: Chien-Kuang

middle: C

last: Ding


title: Using eHi-C to map chromatin loops in mouse beta cells

city: Madison

first: Sunduz

last: Keles

state: WI

address: 1300 University Ave

department: Biostatistics and Medical Informatics, Statistics

institute: University of Wisconsin-Madison

summary: eHiC data of mouse beta cells from C57BL/6 mice

overall_design: Examination of chromatin looping profiles in mouse beta cells from 6 C57BL/6 mice

first: Leina

last: Lu

first: Xiaoxiao

last: Liu

first: Jiajia

last: Xi

first: Fulai

last: Jin

first: Yan


title: RNA-Seq, ChIP-Seq and HiC of Wild Type and CTCF cKO Liver

city: Seoul

first: Hyoung-Pyo

last: Kim

address: Yonsei-ro 50-1

department: Department of Tropical Medicine

institute: Yonsei University College of Medicine


title: Overexpression of homeobox gene DLX1 unravels a novel mechanism in driving prostate cancer

city: Bangalore

first: Genotypic

last: technology

state: Karnataka

address: 259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE

country: India

institute: Genotypic Technology

summary: Higher expression levels of DLX1 has been associated with primary and metastatic prostate tumor. DLX1 along with HOXC6 is well-established diagnostic biomarker for the early detection of prostate cancer (PCa). However, the mechanism involved in DLX1 up-regulation and functional significance in metastatic castration-resistant prostate cancer (mCRPC) progression remains unexplored. Here, we identified that DLX1 serves as an oncogene thereby regulating several oncogenic  properties associated with PCa progression.

overall_design: Agilent two-color experiment,Organism: Human ,Agilent Whole Genome Human 8x60K (AMADID: 072363)

first: Sakshi

last: Goel

first: Vipul

last: Bhatia

first: Mahendra

last: Shyamlal Palecha

first: Shannon

last: Carskadon

first: Nilesh

last: Gupta

first: Nallasivam

last: Palanisamy

first: Bushra

last: Ateeq


title: Genome-wide transcriptional changes induced by Cd2+ in Aspergillus nidulans

city: Debrecen

first: Tamas

last: Emri

address: Egyetem ter 1

country: Hungary

institute: University of Debrecen

summary: Cd2+ pollution represents a serious global environmental risk. Understanding how microbes survive cadmium stress can facilitate the development of techniques to clean our environment and to prevent accumulation of this toxic heavy metal in the food chain. Genome-wide transcriptional changes induced by CdCl2 were determined and evaluated in Aspergillus nidulans. In addition to the reference strain, a atfA gene deletion mutant was also investigated to collect data on the regulatory role of AtfA transcription factor in this model organism. Up-regulation of the crpA Cu2+/Cd2+ pump and AN7729 putative bis(glutathionato)-cadmium transporter genes as well as transcriptional changes aiming to increase intracellular Cys availability were important parts of the efficient adaptation in both strains. Although deletion of atfA did not alter the cadmium tolerance of the fungus, the cadmium stress response of the mutant substantially differed from that of the reference strain. Promoter and transcriptional analyses of the “Two component signal transduction system” genes suggest that the AtfA-dependent regulation of these genes can be relevant in this phenomenon. We concluded that the regulatory network of A. nidulans has a high flexibility allowing the fungus to adapt efficiently to stress both in the presence and absence of this important transcription factor.

overall_design: two strains (TNJ92 atfA gene deletion mutant and THS30 reference strain) were examined under two conditions (CdCl2 treated and untreated cultures) with three biological replicates (altogether 12 samples)

first: Tamás

first: Barnabas

last: Gila

first: Karoly

last: Antal

first: Istvan

last: Pocsi


title: Gene Expression Profiling and Prediction of Survival in Oropharyngeal Tumors

city: Biella

first: Paola

last: Ostano

address: via Malta 3

institute: Fondazione Edo ed Elvo Tempia

summary: Background: Oropharynx squamous cell carcinoma (OPSCC) is a subtype of HNSCC, arising from the base of tongue, lingual tonsil, tonsil, oropharynx, pharynx. The majority of OPSCC positive for HPV infection is associated with better prognosis, but a fraction of them, similarly to HPV-negative ones, is resistant to therapy and has poor prognosis. A deep molecular study of OPSCC is mandatory to identify either prognostic markers or targets for therapy, in particular in patients with worse prognosis.    Methods: 14 HPV-positive and 15 HPV-negative Italian OPSCC (n=29) with complete clinical information and follow-up of more than 5 years were molecularly characterized by gene expression profiling and compared to three cohorts of OPSCC extracted from public HNSCC datasets. AKR1C3 emerged as robust marker overexpressed in HPV-negative OPSCC and in HPV-positive lesions with worse prognosis. Fadu and Cal-27 OPSCC cell lines were treated with AKR1C3 inhibitors, alone or in combination with Cisplatin.  Results: Gene set enrichment analysis revealed that up-regulated genes in HPV-positive samples are involved in immune system, muscle related processes, response to stimuli, actin organization, tissue development and adhesion, while down-regulated genes participated in glutathione derivative biosynthetic and xenobiotic metabolic processes, hypoxia and oxidative stress. AKR1C3, coding for an enzyme involved in chemio-radioresistance, is in the top10 genes with higher upregulation in HPV-negative samples. Pre-treatment with a selective AKR1C3 inhibitor potentiated the effect of Cisplatin in cells with higher basal level of the enzyme.  Conclusions: We identified a druggable target, AKR1C3, associated with survival in subgroups of OPSCC patients either positive or negative for HPV infection and to resistance to chemo-radiotherapy in HNSCC. Pretreatment of OPSCC cell lines expressing this enzyme with a selective AKR1C3 inhibitor is able to enhance Cisplatin efficacy.

overall_design: Gene expression of 29 cases of squamous cell carcinomas of the oropharynx (OPSCC) were profiled with Agilent microarrays. Each sample was analyzed with a dye-swap technical replicate.

first: Caterina

last: Peraldo-Neia

first: Maurizia

last: Mello-Grand

first: Francesca

last: Guana

first: Andrea

last: Carenzo

first: Guido

last: Valente

first: Paolo

last: Aluffi

first: Marco

last: Krengli

first: Gianluca

last: Averono

first: Riccardo

last: Dosdegani

first: Giovanna

last: Chiorino

first: Simonetta

last: Oliaro-Bosso

first: Agnese

last: Pippione

first: Donatella

last: Boschi

last: Bagnasacco


title: Genome-wide chromatin accessibility of founder mice pancreatic islets

first: Sunduz

last: Keles

summary: ATAC-seq data of founder mice pancreatic islets

overall_design: 8 founder strains, 2 sexes, 8 lanes each sample

first: Shane

last: Simonett

first: Donnie

last: Stapleton

first: Kathryn

last: Schueler


title: Metabolomics and Dual RNA-Sequencing on Root Nodules Revealed New Cellular Functions Controlled by Paraburkholderia phymatum NifA

city: Zürich

first: Gabriella

last: Pessi

address: Winterthurerstrasse 190

institute: University of Zürich

summary: Paraburkholderia phymatum STM815 is a nitrogen-fixing endosymbiont that forms root nodules on the agriculturally important Phaseolus vulgaris and other host plants. We previously showed that the nodules induced by a STM815 mutant of the gene encoding the master regulator of nitrogen fixation NifA showed no nitrogenase activity (Fix-) and increased in number compared to P. vul-garis plants infected with the wild-type strain. To further investigate the role of NifA during symbiosis, nodules from P. phymatum wild-type and nifA mutants were collected and analyzed by metabolomics and dual RNA-Sequencing, allowing us to investigate both host and symbiont transcriptome. Using this approach, several metabolites changes could be assigned to bacterial or plant responses. While the amount of the C4-dicarboxylic acid succinate and of several amino acids was lower in Fix- nodules, the level of indole-acetamide (IAM) and brassinosteroids increased in Fix- nodules. Transcriptome analysis identified P. phymatum genes involved in transport of C4-dicarboxylic acids, carbon metabolism, auxin metabolism and stress response to be differen-tially expressed in absence of NifA. Furthermore, P. vulgaris genes involved in autoregulation of nodulation (AON) are repressed in nodules in absence of NifA potentially explaining the hyper-nodulation phenotype of the nifA mutant. These results and additional validation experiments suggest that P. phymatum STM815 NifA is not only important to control expression of nitrogenase and related enzymes but is also involved in regulating its own auxin production and stress re-sponse. Finally, our data indicate that P. vulgaris does sanction the nifA nodules by depleting the local carbon allocation rather than by mounting a strong systemic immune response to the Fix- rhizobia.

sample_organism:
Phaseolus vulgaris
Paraburkholderia phymatum STM815

overall_design: Determination of the NifA regulon during symbiosis using dual-RNASeq and metabolmics

first: Paula

middle: B

last: Sancho

first: Martina

last: Lardi

first: Yilei

last: Liu

first: Leo

last: Eberl

first: Nicola

last: Zamboni

first: Aurélien

last: Bailly


title: In vitro differentiation of hepatoblasts into hepatocytes

status: Public on Jul 26 2021

city: Dresden

first: Lenka

last: Belicova

state: Saxony

address: Pfotenhauerstr. 108

country: Germany

institute: MPI-CBG Dresden

summary: We differentiated primary mouse E14.5 Dlk1+ hepatoblasts in vitro into hepatocyte-like cells that recapitulated morphological features of hepatocytes. To assess the level of differentiation, we performed RNA-seq analysis and compared gene expression profiles of hepatobalsts, in vitro differentiated hepatocytes and mature hepatocytes isolated from adult mouse livers as a control.

overall_design: Hepatoblasts, in vitro differetniated hepatocytes and mature hepatocytes were collected in 4 biological replicates. Hepatoblasts were isolated based on the Dlk1 surface marker using MACS (Tanimizu et al., 2003, JCS). The isolated cells were cultured on fibronectin coated plates in optimized Differentiation media for 4 days (Belicova et al., 2021, JCB). The adult mouse liver hepatocytes were isolated according to a published colagenase perfusion protocol (Klingmüller et al., 2006, IEE Proceedings: Systems Biology).

first: Helin

last: Räägel


title: Founder mice pancreatic islets and beta cells

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Easy-Prime: a machine learning–based prime editor design tool

city: Memphis

first: Yong

last: Cheng

state: Tennessee

address: 262 Danny Thomas Place

department: Department of Hematology

institute: St. Jude Children’s Research Hospital

summary: We developed ML-based methods to optimize PE design

overall_design: PE designs were generated based on Easy-Prime prediction and PrimeDesign recommendation for four target mutations

first: Yichao

last: Li


title: Revealing the molecular mechanism of mouse innate behavior by single-cell sequencing

city: Beijing

first: Zeyuan

address: 15 Datun Road, Chaoyang District, Beijing, 100101, China

institute: Institute of Biophysics,Chinese Academy of Sciences

summary: Innate behaviors, like fear, are basic behaviors which animals possess to ensure their survival and reproduction.  Now we know that in animals, for example mouse, different behaviors are controlled by different or similar neural circuits. Whether these behaviors are coded by specific genes are not clear. In this project, we used single-cell sequencing to solve this problem in fear and hunting behaviors.

overall_design: single cell RNA-seq of the superior colliculus of the adult mouse

first: Mengdi

last: Wang

first: Qian

last: Wu

first: Xiaoqun


title: Transcriptome analysis of CCR9+, CXCR5+ and CCR9-CXCR5- Th cells in primary Sjögren's syndrome (pSS) patients and healthy controls (HC)

city: Utrecht

first: Anneline

last: Hinrichs

address: Postbus 85500

department: Rheumatology & Clinical Immunology

country: Netherlands

institute: University Medical Center Utrecht

summary: CCR9+ Tfh-like pathogenic T helper (Th) cells are elevated in patients with primary Sjögren’s syndrome (pSS) and indicated to play a role in pSS immunopathology.   CCR9+, CXCR5+ and CCR9-CXCR5- (double negative, DN) Th cells from blood of 7 healthy controls (HC) and 7 pSS patients were FACS sorted and RNA sequencing was performed. Our aim is to delineate the CCR9+ Th cell-specific transcriptome to study the molecular dysregulation of these cells in pSS patients. Transcriptomic analysis of circulating CCR9+ Th cells reveals CCR9-specific pathways involved in effector T cell function, equally expressed in pSS patients and HC. Given the increased numbers of CCR9+ Th cells in the blood and inflamed glands of pSS patients and presence of inflammatory stimuli to activate these cells this suggests that CCR9-specific functions, such as cell recruitment upon CCL5 secretion, could significantly contribute to immunopathology in pSS.

overall_design: FACS sorted CCR9+, CXCR5+ and CCR9-CXCR5- Th cells from peripheral blood of n=7 pSS patients and n=7 HC were analyzed using RNA sequencing

first: Aridaman

last: Pandit

first: Joel

last: vanRoon


title: Differential DNA methylation and changing cell type proportions as fibrotic stage progresses in NAFLD

status: Public on Jul 23 2021

city: Atlanta

first: Karen

last: Conneely

state: GA

address: 615 Michael Street

institute: Emory University

summary: Genome wide DNA methylation profiling of normal and NAFLD liver samples. The Illumina Infinium MethylationEPIC BeadChip kit was used to obtain DNA methylation profiles across CpGs.

overall_design: Bisulfite converted DNA from the samples was hybridised to Illumina Infinium MethylationEPIC BeadChip

first: Nicholas

middle: D

last: Johnson

first: Xiumei

first: Christopher

last: Still

first: Xin

last: Chu

first: Anthony

middle: T

last: Petrick

first: Glenn

last: Gerhard

middle: N

first: Johanna

middle: K

last: DiStefano


title: Founder mice islet RNA profiling

first: Sunduz

last: Keles

summary: RNA-seq data of founder mice pancreatic islets

overall_design: 8 founder strains, 2 sexes, with replicates

first: Gary

middle: A

last: Churchill


title: in situ Hi-C in WT and CTCF depleted mouse livers

first: Hyoung-Pyo

last: Kim

summary: Performed in situ Hi-C to analyze higher-order chromatin structure in cKO livers.

overall_design: Examination of in situ Hi-C  in WT and cKO liver. Each sample have 2 replicates.

first: Yeeun

last: Choi

first: Min-Ji

last: Song

first: Woong-Jae

last: Jung


title: Transcriptome analysis of PBMCs reveals distinct immune response in the asymptomatic and re-detectable positive COVID-19 patients

city: Harbin

first: Jinwen

last: Zhang

address: NanGang

institute: College of Bioinformatics

summary: The existence of asymptomatic and re-detectable positive COVID-19 patients presents the disease control challenges of COVID-19. Most studies on immune response of COVID-19 have focused on the moderately or severely symptomatic patients, however little is known about the immune response in asymptomatic and re-detectable positive patients. Here we performed a comprehensive analysis of the transcriptomic profiles of PBMCs from 48 COVID-19 patients.

overall_design: Expression profiles for PBMCs from 48 COVID-19 patients which include 8 asymptomatic, 13 symptomatic, 15 recovering and 12 RP patients.

first: Jiaqi

first: Jianhua

last: Xu


title: Global Analysis of the RNA-Protein Interaction and RNA Secondary Structure Landscapes Identifies Dynamic Changes During Mammalian Erythropoiesis

city: Philadelphia

first: Mengge

last: Shan

state: PA

address: 433 South University Avenue

department: Biology

institute: University of Pennylsvania

summary: In order to investigate the dynamics of RNA secondary structure and changes in RBP-RNA interaction during mammalian erythropoeisis, we performed protein-interaction profile sequencing (PIP-seq) on MEL cells that are 0,2,or 4 days post differentiation

overall_design: 2 replicates per timepoint of protein interaction profile sequencing (PIP-seq) in MEL cells. Each rep is crosslinked with formaldehyde and split into structure-only and protein-bound libraries and then further split based on RNAse treatment (i.e. ssRNAse or dsRNAse)

first: Ian

last: Silverman

first: Xinjun

last: Ji

first: Stephen

last: Liebhaber

first: Brian

last: Gregory


title: Regulated control of gene therapies by drug induced splicing

status: Public on Jul 22 2021

first: Paul

last: Ranum

address: 3501 Civic Center Blvd.

department: Center for Cellular and Molecular Therapy

institute: The Children's Hospital of Philadelphia

summary:
RNA Seq datasets from HEK293 cells treated with 25nM LMI070

To date, gene therapies for human application rely on engineered promoters that cannot be finely controlled.  Here, we report a universal switch element that allows precise control for gene replacement or gene silencing after exposure to a small molecule.  Importantly, these small molecule inducers are in human use, are orally bioavailable when given to animals or humans, and can reach both peripheral tissues and the brain.  Moreover, the switch system (Xon) does not require the co-expression of any regulatory proteins.  Using Xon, translation of desired elements for controlled gene replacement or editing machinery occurs after a single oral dose, and the robustness of expression can be controlled by drug dose and with repeated drug exposure.  The ability of Xon to provide temporal control of protein expression can be adapted to cell biology applications and animal studies. Additionally, due to the oral bioavailability and safety of the drugs employed, the Xon switch provides an unprecedented opportunity to refine gene therapies for more appropriate human application.

overall_design: Data from 4 LMI070 (25nM) treated and 4 DMSO treated HEK293 cell groups were obtained after sequencing across two lanes run on an Illumina Hi-Seq 4000.

first: Alejandro

last: Monteyes

first: Beverly

middle: L

last: Davidson


title: Genome-wide maps of chromatin state in liver.

summary: We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse liver. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.

overall_design: Examination of 3 different histone modifications and 2 different transcription factors in WT and cKO liver.


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and CTCF cKO Transcriptomes

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived liver transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis
Methods: Liver mRNA profiles of 8-week-old wild-type (WT) and liver specific conditional CTCF KO (CTCF cKO) mice were generated by deep sequencing, in quadruplet, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling.
Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: Liver mRNA profiles of 8 weeks old wild type (WT) and CTCF cKO mice


title: Olaparib resistance and cisplatin responses in triple negative breast cancer

city: Detroit

first: Malathy

middle: P

last: Shekhar

state: MI

address: 421 East Canfield Avenue

department: Oncology

institute: Wayne State University

summary: PARP inhibitor and platinum based drugs such as cisplatin are promising therapies for triple negative breast cancer and exploit the deficiencies in BRCA1 or BRCA2, or homologous recombination repair defects. However, PARP inhibitor resistance is proven to be a major clinical problem. Acquired PARP inhibitor resistance has been linked with co-resistance to platinum-based drugs.  To determine how acquired olaparib resistance affects cisplatin response and whether this is influenced by their BRCA1 status, we performed RNAseq transcriptome analysis of isogenic triple negative breast cancer models of olaparib resistance with normal and mutant BRCA1.

overall_design: 54 samples - Parental MDA-MB-468 control (3 samples), Parental MDA-MB-468 Olaparib (3 samples), Parental MDA-MB-468 1 uM cisplatin (3 samples), Parental MDA-MB-468 2 uM cisplatin (3 samples), Parental MDA-MB-468 olaparib + 1 uM cisplatin (3 samples), Parental MDA-MB-468 olaparib + 2 uM cisplatin (3 samples), OlaR MDA-MB-468 15 uM olaparib (3 samples), OlaR MDA-MB-468 15 uM + 1 uM cisplatin (3 samples), OlaR MDA-MB-468 15 uM olaparib + 2 uM cisplatin (3 samples), Parental SUM1315 control (3 samples), Parental SUM1315 Olaparib (3 samples), Parental SUM1315 2 uM cisplatin (3 samples), Parental SUM1315 4 uM cisplatin (3 samples), Parental SUM1315 olaparib + 2 uM cisplatin (3 samples), Parental SUM1315 olaparib + 4 uM cisplatin (3 samples), OlaR SUM1315 25 uM olaparib (3 samples), OlaR SUM1315 25 uM + 2 uM cisplatin (3 samples), OlaR SUM1315 25 uM olaparib + 4 uM cisplatin (3 samples)

first: Shekhar

last: Malathy


title: Sex-specific development in haplodiploid honeybee is controlled by the female-embryo-specific activation of thousands of intronic lncRNAs

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

institute: ABLife, Inc.

summary: Sex determination in the honeybee (Apis mellifera) is governed by the queen-controlled unfertilization or fertilization of embryo, though the mechanisms of determination are poorly understood. Here, we obtained the transcriptomes from individual worker and drone embryo during the embryonic development (day 1 to day 3). We show that transcriptional difference between worker and drone embryo is very small during the first day of hatching, during which sex-determinant gene csd expresses similarly. Differential transcription between worker and drone embryo bursts at day 2, among which csd is induced in worker embryo at day 2 and sex-lethal gene sxl is repressed in male embryo. An unexpected global regulation of alternative splicing accompanies the honeybee embryonic development, and male and worker embryo show distinct regulatory patterns and mechanisms. This study suggests the honeybee sex determination is more globally controlled at both the transcriptional and alternative splicing levels.

overall_design: We collected 2 groups of worker and male (drone) embryo laid by two different honeybee queens. embryo were collected at 1-day (24 hour), 2-day (48 hour) and 3-day (72 hour) after laying. Three worker and male embryo were collected for each time point. A total of 36 embryo were collected for single-embryo RNA-seq.

first: Yi

first: Liming

last: Wu


title: Sequencing of siRNA from soybean containing silencing vectors and empty vectors

status: Public on Jul 21 2021

first: Mehdi

last: Kabbage

state: Wisconsin

address: Department of Plant Pathology, 583 Russell Labs 1630 Linden Dr

department: Department of Plant Pathology

institute: University of Wisconsin Madison

type: Non-coding RNA profiling by high throughput sequencing

summary: A cDNA library was constructed by Novogene (CA, USA) using a Small RNA Sample Pre Kit, and Illumina sequencing was conducted according to company workflow, using 20 million reads. Raw data were filtered for quality as determined by reads with a quality score > 5, reads containing N < 10%, no 5' primer contaminants, and reads with a 3' primer and insert tag. The 3' primer sequence was trimmed and reads with a poly A/T/G/C were removed

overall_design: Examination of siRNA corresponding to Ss-oah1 in S. sclerotiorum in plants containing silencing constructs and plants containing empty vectors

first: Megan

last: McCaghey

first: Dandan

last: Shao

first: Kurcezewski

last: Kurcezewski

first: Ally

last: Lindstrom

first: Ashish

last: Ranjan

first: Steven

last: Whitham

first: Shawn

last: Conley

first: Brett

last: Williams

first: Damon

last: Smith


title: Transcriptome analysis of T cells from mice immunized with Sindbis Virus Spike and/or aOX40

city: NYC

first: Silvana

last: Opp

address: 550 1st Avenue

department: Pathology

institute: NYU School of Medicine

summary: Gene expression analysis of spleenic T cells isolated seven days after mice received  prime vaccine doses of Sindbis.Spike and/or aOX40. The hypothesis tested changes in the transcriptome profiles of isolated T cells from vaccinated and unvaccinated mice.

overall_design: Total RNA was obtained from isolated T cells seven days after prime vaccine regimen compared to naïve (unvaccinated group).

first: Antonella

last: Scaglione

first: Ziyan

last: Lin

first: Daniel

last: Meruelo


title: ChIP-seq analysis to identify transcriptional targets of BMAL1 mammary epithelial cells in culture, and determine if targets of BMAL1 changed upon lactogen induced differentiation of mammary epithelial cells in culture

city: West Lafayette

first: Theresa

last: Casey

state: Indiana

address: 175 S. University Street

department: Animal Sciences

institute: Purdue University

summary: ChIP-seq analysis was used to identify genes potentially regulated by BMAL1 in undifferentiated (UNDIFF) and lactogen hormone differentiated (DIFF) HC11 cultures. HC11 cells are a normal mouse mammary epithelial cell line. Prior to beginning studies, specificity of the ChIP-grade antibody for the BMAL1 protein was confirmed with immunoprecipitation and western blot analysis.  Nanochip analysis of sonicated input DNA indicated that optimal size of sheared fragments was achieved for ChIP-seq analysis.  Moreover, ChIP-qPCR analysis of the promoter region of PER1, a transcriptional target of BMAL1:CLOCK, found that 70% of the input DNA sequence was captured by chromatin immunoprecipitation, with the q-PCR product being 9-fold higher in BMAL1-ChIP samples than mock samples (no primary antibody added). Q-PCR analysis of the exon region of MAGEA 1_2, a sperm specific gene that is not a BMAL1:CLOCK target was used as a negative control.  There was no difference in amount of  MAGEA 1_2 q-PCR between mock-ChIP and BMAL1-ChIP samples, thus further validating the BMAL1-ChIP assay.  The eight samples sequenced for these studies consisted of four pairs of input and ChIP samples with two pairs from UNDIFF and two from DIFF HC11 cultures.  Mapping rate of reads to the mouse genome across the four paired input-ChIP samples averaged 96%.

overall_design: The eight samples sequenced for these studies consisted of four pairs of input and ChIP samples with two pairs from undifferentiated (UNDIFF) and two from lactogen (prolactin, dexamethasone and insulin) differentiated (DIFF) HC11 cultures.

first: Jyothi

last: Thimmapuram


title: Pluripotency-state-dependent Role of Dax1 in Embryonic Stem Cells Self-renewal

city: chongqing

first: wang

last: jiaqi

state: chongqing

address: Gaotanyan Main Street, Shapingba District

institute: army medical school

summary: DAX1 (also known as Nr0b1) is regarded as an important component of the transcription factor network in mouse embryonic stem cells (ESCs). However, the role and the molecular mechanism of Dax1 in the maintenance of different pluripotency states are poorly understood. Here, we constructed a stable Dax1 knockout (KO) cell line using the CRISPR/Cas9 system to analyze the precise function of Dax1. We reported that 2i/LIF-ESCs had significantly lower Dax1 expression than LIF/serum-ESCs. Dax1KO ES cell lines could be established in 2i/LIF and their pluripotency was confirmed. In contrast, Dax1-null ESCs could not be continuously passaged in LIF/serum due to severe differentiation and apoptosis. In LIF/serum, the activities of the Core module and Myc module were significantly reduced, while the PRC2 module was activated after Dax1KO. The expression of most pro-apoptotic genes and lineage-commitment genes were drastically increased, while the down-regulated expression of anti-apoptotic genes and many pluripotency genes was observed. Our research on the pluripotent state-dependent role of Dax1 provides clues to understand the molecular regulation mechanism at different stages of early embryonic development.

overall_design: We established a stable Dax1-knockout (KO) cell line and analyzed the effect of Dax1KO on the cell phenotypes and the pluripotency gene regulatory network under LIF/serum and 2i/LIF conditions, respectively.

first: Junlei

last: Zhang

first: Jianrong

last: He

first: Yan

last: Ruan

first: Jiaqi

last: Wang

first: Chen


title: Single-cell atlas reveals meningeal leukocyte heterogeneity in the developing mouse brain

city: Gothenburg

first: Xiaoyang

address: Medicinaregatan 11

department: Physiology

country: Sweden

institute: University of Gothenburg

summary:
The meninges are important for brain development and pathology. Using single-cell RNA-sequencing, we have generated the first comprehensive transcriptional atlas of neonatal mouse meningeal leukocytes under normal conditions and after perinatal brain injury. We identified almost all known leukocyte subtypes and found differences between neonatal and adult border-associated macrophages, thus highlighting that neonatal border-associated macrophages are functionally immature with regards to immune responses compared to their adult counterparts. We also identified novel meningeal microglia-like populations that appear to participate in white matter development. Early after hypoxic-ischemic insult, neutrophil numbers increased and they exhibited increased granulopoiesis, suggesting that the meninges are an important site of immune cell expansion with implications for the initiation of inflammatory cascades after neonatal brain injury. Our study provides a single-cell-resolution view of the importance of meningeal leukocytes at different stages of development in health and disease.

DOI:10.1101/gad.348190.120

overall_design: Four total meningeal samples, divided in two groups: hypoxic-ischemic (HI) and naïve mice (controls). The samples were processed in two separate runs, each with one HI sample and one naïve sample. All samples were analyzed in terms of gene expression (GEX) and immune profiling (IP) for TCR and BCR clonality,

first: Aura

last: Zelco

first: Vanja

last: Börjesson

first: Jurrian

last: de Kanter

first: Cristina

last: Lebrero-Fernandez

first: Volker

last: Lauschke

first: Eridan

last: Rocha-Ferreira

first: Gisela

last: Nilsson

first: Syam

last: Nair

first: Pernilla

last: Svedin

first: Mats

last: Bemark

first: Henrik

last: Hagberg

first: Carina

last: Mallard

first: Frank

last: Holstege


title: Set1 targets gene with essential identity and tumor suppressing functions in planarian stem cells. Mnase-ChIP was used to validate a wide histone H3K4me3 peak width signature.

city: Lexington

first: Elizabeth

last: Duncan

state: KY

address: 675 Rose St

institute: University of Kentucky

summary: Previous publications have reported the significance of a wide histone H3K4me3 signature at essential genes in many organisms and cell types. Most of these published datasets were produced using cross-linking ChIP-seq, in which the chromatin was chemically cross-linked using formadehyde and then mechanically sheared with sonication. Here we use Mnase-ChIP, a non-cross-linking method that fractionates chromatin using the enzyme micrococcal nuclease, to show that this wide H3K4me3 signature is not a technical artifact of cross-linking ChIP.

sample_organism:
Drosophila melanogaster
Schmidtea mediterranea

overall_design: ChIP-seq was done using an Mnase-based method of chromatin fractionation prior to IP with an antibody to histone H3 K4me3. The experiment was performed using isolated S.mediterannea stem cells (X1 cells) mixed with Drosophila S2 cells; 800K planarian X1 cells were mixed with 9.8M S2 cells, processed for Mnase-ChIP, and the ChIP DNA sequenced with their corresponding input DNA.

first: Prince

last: Verma

first: Court

last: Waterbury


title: Global Analysis of the RNA-Protein Interaction and RNA Secondary Structure Landscapes Identifies Dynamic Changes During Mammalian Erythropoiesis (mRNA)

first: Mengge

last: Shan

summary: Two features of eukaryotic RNA molecules that regulate their post-transcriptional fates are RNA secondary structure and RNA-binding protein (RBP) interaction sites. However, a comprehensive global overview of the dynamic nature of these sequence features during erythropoiesis has never been obtained. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approach to reveal the global landscape of RNA secondary structure and RBP-RNA interaction sites and the dynamics of these features during this important developmental process. We identify dynamic patterns of RNA secondary structure and RBP binding that are consistently anti-correlated throughout mRNAs during erythropoiesis. Additionally, we determine a set of protein-bound sequence motifs and their dynamic structural and RBP binding contexts. Finally, using these dynamically bound sequences, we identify a number of RBPs that have known and putative key functions in post-transcriptional regulation during mammalian erythropoiesis. In total, this global analysis reveals new post-transcriptional regulators of mammalian blood cell development.

overall_design: mRNA-seq of 2 biological replicates of MEL cells at day 0, day 2, and day 4 post differentiation induction by DMSO

first: Xinjun

last: Ji

first: Kevin

last: Janssen

first: Ian

last: Silverman

first: Jesse

last: Humenik

first: Ben

last: Garcia

first: Stephen

last: Liebhaber

first: Brian

last: Gregory


title: Arraystar Human m6A-mRNA & lncRNA Epitranscriptomic microarray analysis in lung adenocarcinoma patient's tissues and adjacent non-tumor tissues

status: Public on Jul 20 2021

city: Shanghai

first: Ping

address: 389 Xincun Road

department: Laboratory Medicine

institute: Shanghai Tongji Hospital, Tongji University School of Medicine

type: Methylation profiling by genome tiling array

summary: Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer and shows high morbidity and mortality rates as well as poor prognosis. However, there is still an urgent need to provide more effective biomarkers for the early diagnosis, prognosis and monitoring of LUAD. The Arraystar Human M6a-MRNA&lncRNA Epitranscriptomic microarray analysis was performed on  six pairs of LUAD tissues and adjacent non-tumor tissues to compare and screen the M6a marker of  LUAD, thus may offer a new avenue of targets and strategies for LUAD diagnosis and treatment.

overall_design: 12 samples were divided into 2 groups:Lung adenocarcinoma patients' tumor tissues（Tumor group） and adjacent non-tumor tissues(Normal group), each containing 6 sample.

first: Dong

last: Li


title: RNA-seq of Human Endometrial B Cells During the Mid-luteal Phase

status: Public on Jul 24 2021

city: Oxford

first: Jen

last: Southcombe

address: John Radcliffe Hospital, Headington, Oxford OX3 9RR

department: Nuffield Department of Women's and Reproductive Health

country: United Kingdom

institute: University of Oxford

summary: We present the transcriptome analysis by RNA sequencing of endometrial B cells identifying potential B cell phenotypes and function in human endometrium during the mid-luteal phase. To achieve this, endometrial B cells were isolated from 15 endometrial biopsies, and cDNA library preparation was performed for RNA sequencing.

overall_design: RNA-seq of 15 endometrial B cell samples (B cells were obtained through CD19+ cell sorting)

first: Mengni

last: Shen

first: Ingrid

last: Granne


title: Transcriptional profile (RNA-seq) of cultured astrocytes [RegionAstro]

city: Neuherberg

first: Pawel

last: Smialowski

address: Ingolstädter Landstraße 1

institute: Helmholtz Zentrum Muenchen

summary: Purpose of the study is to compare the transcriptional profiles of astrocytes obtained from cortex gray matter and spinal cord of postnatal mice.

overall_design: Astrocytes were isolated from 2 regions via ACSA-2 sorting, cultured for 7 days and then subjected to RNA-seq

first: G

last: Masserdotti

first: B

last: Hersbach

first: K

last: Knelles


title: Single cell RNA-seq analysis of astrocyte to neuron reprograming [Patch-seq1]

summary: Purpose of the study is to compare the transcriptional profiles of reprogrammed neurons from spinal cord-derived astrocyte and control astroctes.

overall_design: MACS-sorted astrocytes, obtained from postnatal mouse spinal cord, were transduced in vitro with Ascl1 and Neurog2. Reprogrammed cells were collected from the coverslips by suction and subjected to SmartSeq2 amplification

first: David

last: Petrik


title: Single cell RNA-seq analysis of reprogrammed neurons [Patch-seq2]

summary: Purpose of the study is to compare the transcriptional profiles of reprogrammed neurons from spinal cord-derived astrocytes, analyzed after electrophysiological recordings, and control astroctes.

overall_design: MACS-sorted astrocytes, obtained from postnatal mouse spinalc cord, were transduced in vitro with Ascl1 and Neurog2. Reprogrammed cells were analyzed by electrophysiology ar 3-to-4 weeks, and then single cells were collected and subjected to SmartSeq2 amplification

first: V

last: Bednarova

first: P


title: Next Generation Sequencing analysis of the global expression profiling  in bone marrow derived macrophages response to porcine circovirus type 2.

city: New York

first: Yingli

last: Shang

address: 535E 70th Street

institute: Hospital for Special Surgery

summary: To know the influences of PCV2 infection on macrophage phenotype, we examined the expression profiling of global genes in PCV2 infected-BMDMs by RNA-seq. The results revealed that expression of 369 genes were greater than twofold in PCV2 infected-BMDMs relative to their expression levels in control cells. In addition, the expression of 201 genes were lower than in PCV2-infected macrophages compared to that of control cells . Among these genes, we found that PCV2 infection strikingly induced expression of a subset of M1-associated genes, whereas decreased expression of multiple M2-associated genes, indicating that PCV2 infection likely reprograms the macrophage transcription phenotype.

overall_design: mRNA profiles of BMDMs left uninfected or infected with PCV2 for 12 h.

first: Wen

last: Zhang

first: Zhendong

last: Fu


title: Transcriptomics of THP-1 EVs released after inflammasome or TLR stimulation

city: Melbourne

first: Jamie

last: Gearing

address: 27-31 Wright St

institute: Hudson Institute of Medical Research

summary:
Inflammasome-activated cells undergo an inflammatory cell death associated with the release of potent pro-inflammatory cytokines and poorly characterized extracellular vesicles (EVs). Since inflammasome-induced EVs could signal inflammasome pathway activation in patients with chronic inflammation and modulate bystander cell activation, we performed a systems analysis of the RNA content and function of two EV classes. Therefore, PMA-differentiated THP-1 macrophages were either stimulated with inflammasome activators or TLR ligands and subsequently differently sized EVs (10K and SEC EVs) were isolated from the tissue culture supernatant. The RNA contained within EV preparations was extracted and subjected to the Clariom D microarray.
Web link to Clariom D chip: https://www.thermofisher.com/order/catalog/product/902925?de&en#/902925?de&en

overall_design: Five replicates of each sample were generated, i.e., 110 samples were subjected to a Clariom D Pico microarray. Microarrays were processed in four batches. We removed 17 samples from the analysis due to failed mid-assay quality controls or scan issues. The input RNA amount per sample was 1 ng for 87 samples and < 1 ng for six samples.

first: Christina

middle: F

last: Budden

first: Linden

middle: J

first: Eicke

last: Latz


title: Serum miRNA profiling for early PDAC diagnosis and prognosis: a retrospective study

city: Padova

first: Cristiano

last: De Pitta

address: Via U. Bassi, 58/B

institute: University of Padova

summary: Background: Tumor stage predicts pancreatic cancer (PDAC) prognosis, but prolonged and short survivals have been described in patients with early-stage tumors. Circulating microRNA (miRNA) are an emerging class of suitable biomarkers for PDAC prognosis. Our aim was to identify whether serum miRNA signatures predict survival of early-stage PDAC. Methods: Se-rum RNA from archival 15 stage I-III PDAC patients and 4 controls was used for miRNAs ex-pression profile (Agilent microarrays). PDAC patients with comparable age, gender, diabetes, jaundice and surgery were classified according to survival: less than 14 months (7/15 pts, group A) and more than 22 months (8/15 pts, group B). Bioinformatic data analysis was performed by two-class Significance Analysis of Microarray (SAM) algorithm. Binary logistic regression analyses considering PDAC diagnosis and outcome as dependent variables, and ROC analyses were also performed. Results: 2549 human miRNAs were screened out. At SAM, 76 differen-tially expressed miRNAs were found among controls and PDAC (FDR = 0.4%), the large major-ity (50/76, 66%) of them being downregulated in PDAC with respect to controls. Six miRNAs were independently correlated with early PDAC, and among these, hsa-miR-6821-5p was asso-ciated with the best ROC curve area in distinguishing controls from early PDAC. Among the 71 miRNAs differentially expressed between groups A and B, the most significant were hsa-miR-3135b expressed in group A only, hsa-miR-6126 and hsa-miR-486-5p expressed in group B only. Eight miRNAs were correlated with the presence of lymph-node metastases; among these, hsa-miR-4669 is of potential interest. hsa-miR-4516, increased in PDAC and found as an independent predictor of survival, has among its putative targets a series of gens involved in key pathways of cancer progression and dissemination, such as Wnt and p53 signaling pathways. Conclusions: A series of serum miRNAs was identified as potentially useful for the early diag-nosis of PDAC, and for establishing a prognosis

overall_design: miRNA expression profiles were carried out using the “Agilent SurePrintG3 Human miRNA v.21 (8x60K)” microarrays (Agilent Technologies, Santa Clara, CA, USA)” (Agilent Technologies), that allows the detection of 2.549 known human (miRBase Release 21.0) and 76 viral miRNAs. We analysed expression of circulating miRNAs in 15 PDAC patients as well as 4 reference subjects. An unsupervised hierarchical clustering analysis, by using the list of differentially expressed genes between PDAC patients and controls, enabled the clear separation of controls and PDAC patients with long and short survival, respectively.

first: Ada

last: Aita

first: Caterina

last: Millino

first: Cosimo

last: Sperti

first: Beniamina

last: Pacchioni

first: Mario

last: Plebani

last: De Pittà

first: Daniela

last: Basso


title: Transcriptomics of THP-1 macrophages after treatment with NLRP3-induced EVs

summary: Inflammasome activation in macrophages induces the release of EVs, however, the effect of these inflammasome-induced EVs on recipient cells is poorly characterized. To characterize the effect  EVs released upon LPS + nigericin stimulation, we performed 3' sequencing on the recipient cells (NLRP3 KO THP-1 macrophages and NLRP3 KO THP-1 macrophages that have been reconstituted with NLRP3 to resemble the WT). As controls, RNA isolated from EVs themselves or LPS- or nigericin-treated cells were subjected to 3' sequencing.

overall_design: Five replicaes of each sample were generated, i.e. 100 samples were transferred to the Next Generation Sequencing Core Facility of the Medical Faculty of the University of Bonn for library preparation using the QuantSeq 3’ mRNA Library Prep Kit FWD for Illumina according to the manufacturer’s instructions. Samples were processed in three batches. RNA libraries were prepared with 50 ng of total cellu-lar RNA or 30 ng of total vesicular RNA.  We performed 3’ sequencing on the Illumina Hiseq2500 with an average of 20 · 106 reads per sample.


title: Inflammasome-induced extracellular vesicles harbor distinct RNA signatures and alter bystander macrophage responses

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: RNA Sequencing of HCC cells after lenvatinib, gefitinib, and combination treatment

city: Amsterdam

first: Haojie

last: Jin

address: Plesmanlaan 121

department: The Netherlands Cancer Institute

institute: Division of Molecular Carcinogenesis

summary: Using a kinome-centred CRISPR/Cas9 genetic screen, we identify here that inhibition of the epidermal growth factor receptor (EGFR) is synthetic lethal with lenvatinib in liver cancer cells. We found that the combination of the EGFR inhibitor gefitinib and lenvatinib displays potent anti-proliferative effect in HCC cell lines that express EGFR in vitro and of xenografted HCC cell lines or patient-derived HCC tumours in mice. Herre, we analyzed the different  transcriptome profiling of HCC cells treated with DMSO, lenvatinib, gefitinib, and lenvatinib plus gefitinib by RNA-sequencing.

overall_design: Examination of DMSO, lenvatinib, gefitinib, and lenvatinib plus gefitinib treatment in 4 HCC cell lines.

first: Rene

last: Bernards


title: Sensitivity of cohesin-chromatin association to high salt treatment corroborates non-topological mode of loop extrusion

city: Moscow

first: Arkadiy

last: Golov

address: Vavilova, 34/5

country: Russia

institute: Institute of Gene Biology, RAS

summary: Cohesin is a key organizer of chromatin folding in eukaryotic cells. Two basic activities of this ring-shaped protein complex are maintenance of sister chromatid cohesion and establishment of long-range DNA-DNA interactions through the process of loop extrusion. Though basic principles of both cohesion and loop extrusion have been described we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed stability of cohesin-dependent loops and cohesin association with chromatin in high ionic strength conditions in G1-synchronised HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions which correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. As topologically-engaged cohesin is considered to be salt-resistant, our data corroborate a non-topological model of loop extrusion.

overall_design: We used C-TALE and ChIP-seq to assess sensitivity of cohesin-chromatin association to high salt treatment in HeLa cells


title: m6Am-seq reveals the dynamic m6Am methylation in the human transcriptome

status: Public on Jul 19 2021

city: BeiJing

first: Kai

last: Li

address: Yi He Yuan Street No.5

department: School of Life Science

institute: Peking University

summary: We report m6Am-seq, based on selective in vitro demethylation and RNA immunoprecipitation. m6Am-seq directly distinguishes m6Am and 5’-UTR N6-methyladenosine (m6A).

overall_design: The mAm-seq is divided into a total of three steps: 1) cap-m7G RNA immunoprecipitation; 2) In vitro demethylation treatment; 3) m6A RNA immunoprecipitation. An m6Am peak could be identified by identifying the demethylase sensitivity peak between the FTO(+) and FTO(-) samples.


title: Single-cell transcriptomic analyses of T cells in chronic HCV-infected patients dominated by DAA-induced interferon signaling changes

city: Los Angeles

first: Ana

last: Maretti-Mira

state: CA

address: 1450 Biggy Street, NRT 3514

department: Medicine

institute: USC

summary: Chronic infection with HCV is manifested by dysregulation of innate immune responses and impaired T cell function at multiple levels.  These changes may impact susceptibility to other infections, responsiveness to antiviral therapies, vaccine responsiveness, and development of complications such as hepatocellular carcinoma.  Highly effective direct-acting antiviral (DAA) therapy has revolutionized the management of chronic HCV, with expected cure rates exceeding 95%.  DAA treatment represents a unique opportunity to investigate to what extent elimination of viral replication and chronic antigen stimulation can restore immunologic phenotype. In this study we interrogated the global transcriptional profile of isolated peripheral blood T cells before, during and after IFN-free DAA therapy using single cell mRNA sequencing.  Our results demonstrate that T cells mapped at single-cell resolution have dramatic transcriptomic changes early after initiation of DAA and many of these changes are sustained after completion of DAA therapy.  Specifically, we see a significant reduction in transcripts associated with innate immune activation and interferon signaling such as ISG15, ISG20, IFIT3, OAS and MX1 in many different T cell subsets.  Furthermore, we find an early upregulation of a gene involved in suppression of immune activation, DUSP1, in circulating T cells.  Conclusion:  This study provides the first in-depth transcriptomic analysis at the single-cell level of patients undergoing DAA therapy, demonstrating that IFN-free antiviral therapy in chronic HCV infection induces hitherto unrecognized shifts in innate immune and interferon signaling within T cell populations early, during, and long-term after treatment.  The present study provides a rich data source to explore the effects of DAA treatment on bulk T cells.

overall_design: Six male patients with HCV genotype 1 were prospectively enrolled in this IRB-approved study (University of Southern California HS-18-00254). Blood was collected at baseline, 4 weeks of treatment and 12 weeks after completion of therapy.  All patients experienced a sustained virologic response. Additionaly, blood samples from three non-HCV subjects were collected to serve as a normal controls. T cell enrichment was performed using CD3 microbeads according to the manufacturers protocol (Miltenyi Biotech, Bergisch Gladbach, Germany).  Single-cells suspensions were processed using Chromium Single Cell 3' Reagent Kits v3 for eight of the subjects (including the 3 controls) and v2 for one patient. Libraries were deep sequenced by the USC Molecular Genomics Core, using Illumina NovaSeq6000.

first: Matt

last: Burchill

first: Matthew

last: Salomon

first: Amanda

last: Wieland

first: Lucy

last: Golden-Mason

first: Michael

last: Gale Jr

first: Hugo

last: Rosen


title: Whole Blood Transcriptome Characterization of 3xTg-AD Mouse and its modulation by transcranial Direct Current Stimulation (tDCS)

city: Brescia

first: Chiara

last: Magri

state: Italy

address: Viale Europa

department: DMMT

institute: Università degli Studi di Brescia

summary: The 3xTg-AD mouse is a widely used model in the study of Alzheimer’s Disease (AD). It has been extensively characterized both from the anatomical and behavioral point of view but poorly studied at the transcriptomic level. For the first time, this study characterizes the whole blood transcriptome of the 3xTg-AD mouse at three and six months of age and evaluates how gene expression is modulated by transcranial direct current stimulation (tDCS). RNA-seq analysis revealed 183 differentially expressed genes (DEGs) that were a direct signature of the genetic background of the mouse. The expression profile of age-related genes in the 3 months-old 3xTg-AD mice was more similar to that of 6 months rather than 3 months-control mice, suggesting a premature aging of the 3xTg-AD mice. Moreover, in the 6 months-old 3xTg-AD mice, we observed a high number of DEGs that could represent good peripheral biomarkers of AD progression. Finally, tDCS was associated with gene expression changes in the 3xTg-AD but not in the control mice. In conclusion, this study provides a better molecular characterization of the 3xTg-AD mouse and suggests that blood gene expression can be used to identify new biomarkers of AD progression and treatments effect.

overall_design: Globally, the blood transcriptome of 42 mice was sequenced. We identified 6 sample groups stratified by mouse strain, age and treatment:  three 3-months-old (3 mo.) 3xTg-AD mice;  nine 6-months-old (6 mo.) 3xTg-AD mice; three 3 mo. C57BL/6 wild type (WT) mice; six 6 mo. C57BL/6 WT mice; fourteen 6 mo. 3xTg-AD mice underwent tDCS stimulation; seven 6 mo. C57BL/6 WT mice underwent tDCS stimulation

first: Massimo

last: Gennarelli

first: Erika

last: Vitali

first: Sara

last: Sacco

first: Edoardo

last: Giacopuzzi

first: Marco

last: Rinaudo

first: Paolo

last: Martini

first: Alessandro

last: Barbon

first: Claudio

last: Grassi


title: CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming

city: Houston

first: Eveline

last: Barbieri

state: Texas

address: 1102 Bates Street

department: Department of Pediatrics

institute: Baylor College of Medicine

summary: Neuroblastoma (NB) arises from neural crest cells (NCCs) secondary to a block in differentiation. Retinoic acid (RA) differentiation therapy has limited therapeutic efficacy, and the mechanisms preventing terminal differentiation remain elusive. We found that the chromatin modifier CHAF1A restricts neuronal differentiation and promotes NB oncogenesis. CHAF1A blocks NC differentiation into mature neurons in both human NCCs and zebrafish models. CHAF1A gain-of-function promotes cell malignancy, blocks RA-induced differentiation, and is sufficient to induce NB tumor formation. Mechanistically, CHAF1A blocks NB differentiation by repressing gene expression programs promoting neuronal development and differentiation, and rewiring polyamine metabolism. Targeting polyamine synthesis restores NB sensitivity to RA therapy. Our results demonstrate that CHAF1A critically contributes to NB oncogenesis by blocking neuronal differentiation and reprogramming cell metabolism.

overall_design: To address the mechanisms through which CHAF1A promotes oncogenesis, we carried out gene expression profiling of SHEP cells upon conditional CHAF1A overexpression over time (0, 24, 72, and 96 h). Total RNA was extracted and processed by the random primed RT-IVT-RT method using the GeneChip WT cDNA Synthesis and Amplification Kit (Affymetrix, Santa Clara CA). cDNA was synthesized from 200 ng of total RNA by reverse transcription using T7 promoter-(N6) oligonucleotides as primers. cDNA was then fragmented and hybridized for 17 h at 45oC to GeneChip® Human Gene 2.0 ST Arrays (Affymetrix). Raw microarray CEL files were processed in R (3.4.1) using the biocLite Affy package (Gautier et al., 2004) function rma and the hugene.2.0st annotation.

first: Ling

last: Tao


title: RGS5 channels intracellular signaling pathways to feature growth arrest of vascular smooth muscle cells

city: Mannheim

first: Carsten

last: Sticht

address: Theodor-Kutzer-Ufer

department: ZMF

institute: University Heidelberg

summary: Members of the regulator of G-protein signaling (RGS) family terminate the activity of specific Ga subunits. As such, RGS5 acts as an inhibitor of Gαq/11 and Gαi/o activity in vascular smooth muscle cells (VSMCs), which regulate the arterial tone and blood pressure. While Rgs5 shows a variable expression in different types of mouse arteries, neither global nor SMC-specific genetic ablation of Rgs5 altered the blood pressure. To study the impact of RGS5 on the VSMC phenotype and signaling, Rgs5 expression was silenced by siRNA in 3D spheroids supporting a contractile VSMC phenotype. Loss of RGS5 elevated the baseline calcium level and the agonist-induced Gαq/11-mediated release of calcium but inhibited RhoA activity. In contrast, overexpression of RGS5 in 2D-cultured proliferating VSMCs promotes their resting state as evidenced by gene expression array-based analyses and attenuated the activity of Akt- and MAP kinase-related signaling cascades. Moreover, RGS5 overexpression attenuated ERK1/2 phosphorylation, VSMC proliferation and migration, which was mimicked by selectively inhibiting Gαi/o but not Gαq/11 activity.  Collectively, the heterogeneous expression level of Rgs5 suggests artery type-specific functions comprising the inhibition of acute agonist-induced Gαq/11/calcium-mediated VSMC contraction as well as the support of a resting VSMC phenotype with low ERK1/2 activity by chronically suppressing the activity of Gαi/o.

overall_design: 3 samples of AdGFP- and 3 samples of AdRGS5-treated HUASMCs were processed for RNA extraction.

first: Thomas

last: Korff


title: MultiK: An Automated Tool to Infer Optimal Cluster Numbers in Single-Cell RNA Sequencing Data

city: Chapel Hill

first: Charles

middle: M.

last: Perou

address: 12-044 Lineberger Comprehensive Cancer Center CB# 7295

department: Professor of Genetics, and Pathology & Laboratory Medicine; Lineberger Comprehensive Cancer Center

institute: University of North Carolina at Chapel Hill

summary: This paper proposes MultiK, a data-driven tool for objective selection of multiple insightful numbers of clusters (K) from single-cell RNA-seq data.

overall_design: MultiK combines multiple clustering resolution solutions together through a consensus approach,  and is capable of identifying robust and reproducible groups across multiple datasets.

first: Aatish

last: Thennavan


title: Early induction of neurogenic program by Ascl1ERT2 and Neurog2ERT2

first: Pawel

last: Smialowski

summary: Spinal cord-derived astrocytes underwent direct neuronal reprogramming following the activation of Ascl1ERT2 and Neurog2ERT2 for 24 hours

overall_design: Spinal cord-derived astrocytes were transduced in cultures with retrovirus enconding a tamoxifen-dependent form of Ascl1 and Neurog2.Cells were treated for 24 hours, then transduced cells were FACS-sorted and their transcriptome analyzed.

first: G

last: Masserdotti

first: K

last: Knelles


title: Depletion of m6A writers and readers impair 3'-end formation in Toxoplasma

city: Indianapolis

first: Michael

middle: James

last: Holmes

state: IN

address: 635 Barnhill Drive

department: Pharmacology & Toxicology

institute: Indiana University School of Medicine

summary: Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3’-boundary of mRNAs within the consensus sequence, YGCAUGCR. Knockdown of the m6A writer components METTL3 and WTAP resulted in diminished m6A marks and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3’-end processing of pre-mRNAs. Loss of METTL3, WTAP, or YTH1 lead to a defect in transcript 3’-end formation. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3’-ends.

overall_design: expression changes in Toxoplasma upon IAA-induced knockdown of METTL3, WTAP, and YTH1 for 4 and 16 h

first: William

last: Sullivan Jr


title: Marginating Transitional B cells Modulate Neutrophils in the Lung During Inflammation and Pneumonia

city: Calgary

first: Jeff

last: Biernaskie

state: Alberta

address: 3330 Hospital Drive NW

department: Comparative Biology and Experimental Medicine

country: Canada

institute: University of Calgary

summary: Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM+ B cells in terms of regulatory properties. Using single cell RNA sequencing, we discovered eight IgM+ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 (T1B) and 2 (T2B) B cells. Lung intravital confocal microscopy revealed that T2B marginate in the pulmonary capillaries via CD49e and required CXCL13 and CXCR5. During lung inflammation, marginated T2B dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A4 (LXA4). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells and LXA4 recapitulated neutrophil regulation in B-cell deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM+ B cell subsets with marginating capillary T2B that dampen neutrophil responses.

overall_design: Intact lungs were dissected from 7 mice (all females) and placed in FACS tube with 4 ml of 1X PBS. A gentleMACS™ Dissociator 2.01 was used to mechanically liberate lung cells and cell suspension was filtered through a 70-μm cell strainer. Cell strainer was rinsed with an additional 6 ml of 1X PBS to recover remaining cells. Tissue samples were centrifuged at 2000 rpm for 5 min and supernatant was discarded. To eliminate erythrocyte contamination in single cell suspension, 2 mL of red blood cell (RBC) lysis buffer was added for 3 min. RBC lysis buffer was diluted by adding 10 ml of PBS. The resulting cell suspension was centrifuged at 2000 rpm for 5 min and supernatant containing the medium plus enzyme was discarded. Cells were resuspended in 500 μL of PBS and 12.5 μL of Fc Block was added for 15 min to block Fc receptors before being exposed to anti-CD45-PeCy7 antibody. Following another round of centrifugation at 2000 rpm for 5 min, cell pellet was first stained with 7-AAD viability dye (5μL per 200 μL) for 7 minutes in dark and then with 1 μL of anti-CD45-PeCy7 in 200 μl of final volume for 20 min. Unbound antibodies were rinsed off by centrifuging cells at 2000 rpm for 5 min and discarding the supernatant. Cells were resuspended in 300μL of PBS + 2% BSA then filtered through a 100-μm cell strainer. 7AAD- CD45+ viable immune cells were sorted in PBS + 2% BSA. Percentage of CD45+ cells was determined by flow cytometry and varied between 92-97%. Following centrifugation at 2000 rpm for 10 min, 40,000 CD45+ single cells were resuspended in a 50 μl of PBS + 2% BSA + 1 μl of RNAse inhibitor solution.

first: John

last: Podstawka

first: Sarthak

last: Sinha

first: Carlos

last: Hiroki

first: Nicole

last: Sarden

first: Elise

last: Granton

first: Elodie

last: Labit

first: Jung

middle: H

last: Kim

first: Graciela

last: Andonegui

first: Yuefei

last: Lou

first: Brendan

last: Snarr

first: Donald

last: Sheppard

last: Rosin

first: Bryan

middle: G

last: Yipp


title: Placental transcriptome adaptations to maternal nutrient restriction in sheep

status: Public on Jul 17 2021

city: College Station

middle: Carey

last: Satterfield

state: TX

address: 2471 TAMUS

department: Animal Science

institute: Texas A&M University

summary: Placental development is modified in response to maternal nutrient restriction (NR) resulting in a spectrum of fetal growth rates. Pregnant sheep carrying singleton fetuses and fed either 100% (n=8) or 50% (NR; n=28) of their National Research Council (NRC) recommended intake from Days 35 to 135 of pregnancy were used to elucidate placentome transcriptome alterations at both Day 70 and 135. NR fetuses were further designated into upper (NR NonSGA; n=7) and lower quartiles (NR SGA; n=7) based on Day 135 fetal weight. At Day 70 of pregnancy, there were 22 genes dysregulated between NR SGA and 100% NRC placentomes, 27 genes between NR NonSGA and 100% NRC placentomes, and 22 genes between NR SGA and NR NonSGA placentomes. These genes mediated molecular functions such as MHC class II protein binding, signaling receptor binding, and cytokine activity. Gene Set Enrichment Analysis (GSEA) revealed significant overrepresentation of genes for natural killer cell-mediated cytotoxicity in NR SGA compared to 100% NRC placentomes and alterations in nutrient utilization pathways between NR SGA and NR NonSGA placentomes at Day 70. Results identify novel factors associated with impaired function in SGA placentomes and potential for placentomes from NR NonSGA pregnancies to adapt to nutritional hardship.

overall_design:
Samples from control pregnancies (n=8) as well as the upper (n=7) and lower (n=7) quartiles of the nutrient restricted pregnancies were analyzed at Days 70 and 135 of pregnancy.

***Please note that raw data are not provided because they were lost***


title: Therapeutic manipulation of SRSF1 mitigates genome-wide transcriptome alterations and neuronal hyperexcitability in C9ORF72-linked amyotrophic lateral sclerosis

city: Napoli

first: Ilaria

last: Granata

address: Via Pietro Catellino, 111

department: Institute for high performance computing and networking (ICAR)

institute: National Research Council of Italy (CNR)

summary: Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to relentless paralysis and death usually within a few years from symptom onset. Thousands of RNA molecules with roles in multiple cellular pathways are compromised in disease challenging the identification of alterations causing pathogenesis over downstream changes consequent to neurodegeneration. We recently showed that partial depletion of SR-rich splicing factor SRSF1 inhibits the nuclear export of pathological C9ORF72-repeat transcripts and subsequent translation of dipeptide-repeat proteins in patient-derived neurons and Drosophila, providing in turn a promising strategy of neuroprotection for the most common form of ALS. While the roles of SRSF1 remain poorly characterised in neurons, its therapeutic manipulation offers a rare opportunity to identify disease-modifying gene expression changes. Here, we report diseased and neuroprotected transcriptomes from human-derived neurons and Drosophila heads. Strikingly, while the depletion of SRSF1 has limited effects on genome-wide expression, splicing and nuclear export of RNAs, reversal of 90 disease-modifying transcripts were identified over 2,000 RNA changes in human disease. Functional validation further demonstrated that conserved potassium intermediate/small conductance calcium-activated KCNN channel inhibitors mitigate C9ORF72-ALS mediated hyperexcitability and death of human motor neurons as well as Drosophila motor deficits. Our data support efficacy and safety of SRSF1 manipulation as a therapeutic approach leading to rescue of multiple biological processes without disrupting neuronal transcriptomes. In addition to a potential new therapeutic target for pharmacological inhibition, we also identified in vitro and in vivo disease-modifying gene expression signatures to guide the development of new biomarkers and novel therapeutic approaches in C9ORF72-ALS/FTD.

overall_design: A total of 24 mRNA profiles, were sequenced using Illumina Illumina HiSeq 4000 (Paired-end, 2x150 bp sequencing, generating data from >280 M clusters per lane). 3 Whole-cell transcriptomes (WCT) and 3 cytoplasmic transcriptomes (CyT) were rofiles for both healthy control (H) and for  C9ORF72-ALS (C9) patient-derived neurons treated with Ctrl-RNAi and SRSF1-RNAi lentivirus. A total of 12 samples for each group were sequenced with a total of three biological replicates.

first: Marta

last: Milo

first: Guillaume

middle: M

last: Hautbergue


title: Regulation of NFAT5/TonEBP-mediated transcriptional responses of cholesterol-exposed vascuar smooth muscle cells

first: Carolina

last: Delatorre

address: Theodor-Kutzer-Ufer 1-3

summary: Objective: To study the impact of NFAT5 overexpression on the transcriptome of cholesterol-exposed murine aortic smooth muscle cells.

overall_design: 4 samples of AdPl- and 4 samples of AdN5-treated VSMCs were processed for RNA extraction

first: Thomas

last: Korff


title: Intra-tumoral activation of endosomal TLR pathways reveals a distinct role for TLR3 agonist dependent type-1 interferons in shaping the tumor immune microenvironment

status: Public on Jul 16 2021

city: San Diego

first: Wenjing

last: Yanf

address: 10770 Science Center Drive

department: Oncology

institute: Pfizer

summary: Toll-like receptor (TLR) agonists have received considerable attention as therapeutic targets for cancer immunotherapy owing to their ability to convert immunosuppressive tumor microenvironments towards a more T-cell inflamed phenotype. However, TLRs differ in their cell expression profiles and intracellular signaling pathways, raising the possibility that distinct TLRs differentially influence the tumor immune microenvironment.

overall_design: Using single-cell RNA-sequencing, we address this by comparing the tumor immune composition of B16F10 melanoma following treatment with agonists of TLR3, TLR7, and TLR9. Marked differences are observed between treatments, including decreased tumor-associated macrophages upon TLR7 agonist treatment.

last: Yang

first: Graham

last: Thomas


title: Copy number variation and expression of exportin-4 associates with severity of fibrosis in metabolic associated fatty liver disease

city: Sydney

first: Mayada

last: Metwally

state: NSW

address: 176 Hawkesbury Rd , Westmead

institute: WIMR

summary: A duplication in the exportin 4 (XPO4) CNV reduces the expression of hepatic XPO4 leading to maintaining sustained activation of SMAD3/SMAD4 and liver fibrosis.

overall_design: We overexpressed Xpo4 in JS-1 cells with and without treatment of TGFβ and undertook RNA-Seq analysis and compared to empty vector control

first: M

first: A

last: Bayoumi

last: Khan

first: L

last: Adams

first: R

last: Aller

first: C

last: García-Monzón

last: Arias-Loste

first: E

last: Bugianesi

last: Miele

last: Anna

first: O

last: Latchoumanin

first: S

last: Han

last: Alenizi

last: EL Sharkawy

last: Elattar

last: Gallego-Durán

first: J

last: Fischer

first: T

last: Berg

last: Liddle

last: Romero-Gomez

last: George

last: Eslam


title: Satellite cell depletion results in a cryptic subpopulation of myonuclei in response to exercise

city: Lexington

first: Yuan

last: Wen

state: Kentucky

address: 760 Press Ave

institute: University of Kentucky

summary: Skeletal muscle is composed of post-mitotic myofibers that form a syncytium containing hundreds of myonuclei. Using a progressive exercise training model in the mouse and single nucleus RNA-sequencing (snRNA-seq) for high resolution characterization of myonuclear transcription, we show myonuclear functional specialization in muscle from sedentary mice that is altered by 4 weeks of exercise training. Furthermore, in response to exercise, snRNA-seq reveals that resident muscle stem cells, satellite cells, were activated, but demonstrated limited lineage progression while contributing to muscle adaption. In the absence of satellite cells, trajectory analysis predicts the emergence of cryptic myonuclei that appear to lose their canonical identity. These data illustrate how myonuclear adaptation to exercise may be disrupted under conditions satellite cell compromise.

overall_design: We used a genetic mouse model to fluorescently label and purify myonuclei from skeleletal muscle to investigate single-myonuclear transcriptomes using single-nucleus RNA-sequencing. Additionally, we used a translatable model of exercise training in conjunction with a mouse model of inducible satellite cell (skeletal muscle precursor cells) ablation to study single-nuclear transcriptomic changes after 4-weeks of exercise training at 4 and 24 hours after the last bout of exercise.

first: Davis

last: Englund

first: John

last: McCarthy

first: Charlotte

last: Peterson


title: single-cell RNAseq data from 10-week-old C57BL/6 mice

city: Nanjing

first: Yuchao

last: Jing

state: Jiangsu

address: 101 Longmian Ave Jiangning District

department: Immunology

institute: Nanjing Medical University

summary: The aim of this study is to characterize the testicular immune microenvironment of normal conditional mice

overall_design: 10000 testicular  immune  cells  were sorted from 10-week-old C57BL/6  mice and the single-cell suspension was loaded into a BD Rhapsody cartridge with >200,000 microwells, and single-cell capture was achieved by random distribution and gravity precipitation.Sequencing libraries were prepared using random priming PCR

first: Xiaoming

last: Wang